U.S. patent number 5,766,841 [Application Number 08/479,789] was granted by the patent office on 1998-06-16 for kit for visually detecting the presence of a clinical specimen.
This patent grant is currently assigned to Oklahoma Medical Research Foundation. Invention is credited to C. Worth Clinkscales, Avraham Liav, James F. Maher, Michael D. Roark, Craig D. Shimasaki.
United States Patent |
5,766,841 |
Liav , et al. |
June 16, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
Kit for visually detecting the presence of a clinical specimen
Abstract
A kit for visually detecting the presence of a virus in a direct
clinical specimen is provided. The kit employs a rapid and direct
assay for visually detecting a virus having a characteristic enzyme
in a direct clinical sample without the need for any culturing
step, This assay contains the following steps: (1) the clinical
sample is contacted in solution with a substrate for the enzyme
which includes a chromogen that is cleaved from the substrate by
the enzyme and a precipitating agent which reacts with the
liberated chromogen to form a precipitate, (2) the precipitate is
concentrated, and (3) the concentrated precipitate is visually
observed for the characteristic color of the chromogen. The
observance of the characteristic color confirms the presence of the
virus in the clinical specimen. The kit contains (a) a buffer
solution for extraction of the characteristic enzyme from the
clinical sample; (b) a substrate for the characteristic enzyme,
where the substrate contains a chromogen that is liberated by
reaction between the characteristic enzyme and the substrate, if
the characteristic enzyme is present in the clinical sample; (c) a
precipitating agent which reacts with the liberated chromogen in
the buffer solution to produce a colored precipitate if the
characteristic enzyme is present in the clinical sample; and (d) a
device for concentrating the colored precipitate sufficiently to
provide a distinct, visibly detectable spot of the colored
precipitate if the characteristic enzyme is present in the clinical
sample.
Inventors: |
Liav; Avraham (Denver, CO),
Shimasaki; Craig D. (Tulsa, OK), Maher; James F. (Broken
Arrow, OK), Clinkscales; C. Worth (Tulsa, OK), Roark;
Michael D. (Owasso, OK) |
Assignee: |
Oklahoma Medical Research
Foundation (Oklahoma City, OK)
|
Family
ID: |
27092469 |
Appl.
No.: |
08/479,789 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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133605 |
Oct 8, 1993 |
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963505 |
Oct 16, 1992 |
5252458 |
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635849 |
Dec 31, 1990 |
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Current U.S.
Class: |
435/5; 435/18;
435/975; 435/810 |
Current CPC
Class: |
C12Q
1/04 (20130101); Y10S 435/81 (20130101); Y10S
435/975 (20130101); C12Q 2334/70 (20130101); C12Q
2334/30 (20130101) |
Current International
Class: |
C12Q
1/04 (20060101); C12Q 001/70 () |
Field of
Search: |
;435/5,18,810,975
;436/165 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 269 876 |
|
Jun 1988 |
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EP |
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0 269 876 A12 |
|
Jun 1988 |
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EP |
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Other References
Yolken, R., Fluorometric Assay for Measurement of Viral
Neuraminidase--Application to the Rapid Detection of Influenza
Virus in Nasal Wash Specimens, J of Infectious Diseases 142(4)
516-523, Oct. 1980. .
Pachucki, "Early Detection of Influenza Virus by Using a
Fluorometric Assay of Infected Tissue Culture", J. of Clinical
Microbiology, vol. 26, No. 12 (Dec. 1988) pp. 2664-2666. .
Yolken et al., "Fluorometric Assay for Measurement of Viral
Neuraminidase--Application to the Rapid Detection of Influenza
Virus in Nasal Wash Specimens", J. of Infectious Diseases, No. 4,
(Oct. 1980) pp. 516-523. .
Zbiral et al., "Synthesis of the 4-Methylumbelliferyl
2.alpha.-Glycosides of 7-Epi, 8-Epi-, and 7,8-Bis (epi)
-N-acetylneuraminic Acids, as well as of the 7-Deoxy-, 8-Deoxy,
9-Deoxy, and 4,7-Dideoxy-N-acetylneuraminic Acids and Their
Behaviour Towards Sialidase from Vibrio cholerae", Liebigs, Ann.
Chem. (1989) pp. 519-526. .
Zbiral et al., Structural Transformations of N-Acetylneuraminic
Acid, VII. Synthesis of 7-, 8-, 9-Desoxy-and 4,
7-Didesoxyneuraminic Acid, Monatshefte fur Chemie, vol. 119 (1988)
pp. 127-141. .
Kim et al., "Enzymes in Carbohydrate Synthesis: N-Acetylneuraminic
Acid Aldolase Catalyzed Reactions and Preparation of
N-Acetyl-2-deoxy-D-neuraminic Acid Derivatives"J. Am. Chem. Soc.,
vol. 110 (1988), pp. 6481-6486. .
Kiyoyani et al., "Enzmological Characteristics of Avian Influenza A
Virus Neuraminidase" Microbiol. Immunol., vol. 31, No. 11 (1987)
pp. 1131-1135. .
Kiyotani et al., Enzymological Heterogeneity of Influenza B Virus
Neuraminidase Demostrated by the Flurometric Assay Method, Zbl.
Bakt. Hyq., vol. (A260) (1985) pp. 273-285. .
Kiyotani et al., "Fluorometric Measurement of Neuraminidase
Activity of Influenza Viruses", Hiroshima J. of Medical Sciences,
vol. 33, No. 2, pp. 287-292. .
Yolken, "Enzyme Immunoassays for the Detection of Infectious
Antigens in Body Fluids: Current Limitations and Future Prospects",
Reviews of Infectious Diseases, vol. 4, No. 1 (1982) pp. 35-68.
.
Yolken, "Enzymic Analysis for Rapid Detection of Microbial
Infection in Human Body Fluids: An Overview", Clinical Chem., vol.
27, No. 9 (1981), pp. 1490-1498. .
Myers et al. "The Synthesis of 4-Methylumbelliferyl
.alpha.-Ketoside of N-Acetylneuraminic Acid and Its Use in a
Fluorometric Assay for Neuraminidase", Analytical Biochem., vol.
101, (1980), pp. 166-174. .
Santer, "A Rapid Assay for Neuraminidase--the Detection of Two
Differences in Acivity Associated With Virus Transformation",
Biochimica et Biophysica Acta, vol. 523, (1978) pp. 435-442. .
Beau, "Metabolism of 4-O-Methyl-N-acetylneuraminic Acid a Synthetic
Sialic Acid", 1 Eur. J. Biochem., vol. 106, (1980), pp. 4279-4283.
.
Gross et al. Interaction of N-Acetyl-4-epi-D-neuraminic Acid with
Key Enzymes of Sialic Acid Metabolism, Biochemistry, vol. 27,
(1988) pp. 4279-4283. .
Hagedorn et al., "Synthesis and Biological Properties of
N-Acetyl-4-deoxy-D-neuraminic Acid" Helvetica Chimca Acta, vol. 69
(1986) pp. 2127-2133. .
Baumberger et al., "4-Methylumbelliferyl
5-Acetamido-3,4,5-trideoxy-.alpha.-D-manno-2-nonulopyranosidonic
Acid: Synthesis and Resistance to Bacterial Sialidases", Helvetica
Chimica Acta, vol. 69, (1986), pp. 2127-2133. .
Gross et al. N-Acetyl-4-deoxy-D-neuraminic Acid is Activated and
Transferred on to Asialoglycoprotein, Glyco Conjugate, vol. 4,
(1987) pp. 145-156. .
Bobrow et al., "Catalyzed reporter Deposition, A Novel Method of
Signal Amplification" J. Immunol. Methods, 125 (1989) pp.
279-285..
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Primary Examiner: Gitomer; Ralph
Attorney, Agent or Firm: Fitch, Even, Tabin &
Flannery
Parent Case Text
This application is a continuation of application Ser. No.
08/133,605, filed Oct. 8, 1993, now abandoned, which is a
Divisional of U.S. application Ser. No. 07/963,505, filed Oct. 16,
1992, now U.S. Pat. No. 5,252,458, which is a Continuation of U.S.
application Ser. No. 07/635,849, filed Dec. 31, 1990, now
abandoned.
Claims
We claim:
1. A kit for the detection of the presence of a virus in a direct
clinical sample suspected of containing the virus, wherein the
virus has a characteristic enzyme, said kit comprising;
(a) a buffer solution for extraction of the characteristic enzyme
from the direct clinical sample;
(b) a substrate for the characteristic enzyme, said substrate
comprising a chromogen that is liberated upon incubation of the
direct clinical sample with the substrate by reaction between the
characteristic enzyme and the substrate, if the characteristic
enzyme is present in the direct clinical sample;
(c) a precipitating agent which reacts with the liberated chromogen
in the buffer solution to produce a colored precipitate if the
characteristic enzyme is present in the direct clinical sample;
and
(d) a means for concentrating the colored precipitate sufficiently
to provide a distinct, visibly detectable spot of the colored
precipitate if the characteristic enzyme is present in the direct
clinical sample;
wherein the virus is selected from the group consisting of
influenza A, influenza B, adenovirus, cytomegalovirus, herpes
simplex, parainfluenza, and respiratory synctical virus.
2. The kit of claim 1 wherein the means for concentrating comprises
a porous membrane having a pore size capable of retaining the
precipitate and an absorbent pad beneath and in contact with the
membrane, said pad being capable of wicking away fluid from the
membrane.
3. The kit of claim 2 wherein the pore size is from about 0.2
microns to about 20 microns.
4. The kit of claim 3 wherein the pore size is from about 0.45
microns to about 5 microns.
5. The kit of claim 2 wherein the concentration means further
comprises a focusing device to focus the precipitate on a small
spot on the porous membrane.
6. The kit of claim 5 wherein the small spot upon which the
precipitate is focused is about 1 to 5 mm in diameter.
7. The kit of claim 1 further comprising at least one control
specimen selected from the group consisting of a positive control
specimen and a negative control specimen.
8. The kit of claim 1 wherein the chromogen is selected from the
group consisting of 3-methoxyphenol, 3-dimethylaminophenol,
6-bromo-2-naphthol, 4-chloro-1-naphthol, 3-bromophenol, and
3-acetamidophenol.
9. The kit of claim 1 wherein the precipitating agent is selected
from the group consisting of Fast Red ITR, Fast Red AL, Fast Violet
B, Fast Red-Violet LB, Fast Black K, Fast Blue BB, and Fast Corinth
V.
10. The kit of claim 1 wherein the virus is influenza virus and the
enzyme is neuraminidase.
11. A kit for detecting the presence of an influenza virus in a
direct clinical sample suspected of containing the influenza virus,
wherein the influenza virus has a characteristic neuraminidase
enzyme and wherein the direct clinical sample is collected by means
of a nasopharyngeal swab, a throat swab, a throat wash, a nasal
wash, a nasopharyngeal wash, or a nasopharyngeal aspirate, said kit
comprising;
(a) a buffer solution for extraction of the characteristic
neuraminidase enzyme from the direct clinical sample;
(b) a substrate for the characteristic neuraminidase enzyme, said
substrate comprising a chromogen that is liberated upon incubation
of the direct clinical sample with the substrate by reaction
between the characteristic neuraminidase enzyme and the substrate,
if the characteristic neuraminidase enzyme is present in the direct
clinical sample;
(c) a precipitating agent which will react with the liberated
chromogen in the buffer solution to produce a colored precipitate
if the characteristic neuraminidase enzyme is present in the direct
clinical sample; and
(d) a means for concentrating the colored precipitate sufficiently
to provide a distinct, visibly detectable spot of the colored
precipitate if the characteristic neuraminidase enzyme is present
in the direct clinical sample.
12. The kit of claim 11 wherein the means for concentrating
comprises a porous membrane having a pore size capable of retaining
the precipitate and an absorbent pad beneath and in contact with
the membrane, said pad being capable of wicking away fluid from the
membrane.
13. The kit of claim 12 wherein the pore size is from about 0.2
microns to about 20 microns.
14. The kit of claim 13 wherein the pore size is from about 0.45
microns to about 5 microns.
15. The kit of claim 12 wherein the concentration means further
comprises a means to focus the precipitate on a small spot on the
porous membrane.
16. The kit of claim 15 wherein the small spot upon which the
precipitate is focused is about 1 to 5 mm in diameter.
17. The kit of claim 11 further comprising positive and negative
control specimens.
18. The kit of claim 11 wherein the chromogen is selected from the
group consisting of 3-methoxyphenol, 3-dimethylaminophenol,
6-bromo-2-napththol, 4-chloro-1-naphthol, 3-bromophenol, and
3-acetamidophenol.
19. The kit of claim 11 wherein the precipitating agent is selected
from the group consisting of Fast Red ITR, Fast Red AL, Fast Violet
B, Fast Red-Violet LB, Fast Black K, Fast Blue BB, and Fast Corinth
V.
Description
FIELD OF THE INVENTION
This invention relates to diagnostic tests for detection of viruses
in clinical specimens. More specifically it relates to visual
detection of the presence of a virus that has a characteristic
enzyme through the reaction of the enzyme with a chromogenic
substrate for the enzyme and an agent that reacts with the
liberated chromogen to produce a visually detectable product.
BACKGROUND OF THE INVENTION
The rapid diagnosis of viral infections is becoming an integral
part of good medical practice. Some viruses have definable antigens
against which antibodies can be produced. Therefore, immunoassays
have been widely used for the measurement of the antigen and thus
the determination of the presence or absence of a virion. There are
cases, however, where antibody-antigen reactions are too specific,
and the test may not reliably diagnose a viral infection. Where it
is desirable to measure a more broad group of virions, it may be
possible to detect a particular structural component of the virus.
For example, influenza viruses possess surface glycoproteins that
have neuraminidase activity. The surface glycoproteins hydrolyze
substrates that contain .alpha.-ketosidically linked
N-acetylneuraminic acid. When a virion with neuraminidase activity
is incubated with a chromogenically or fluorometrically modified
N-acetylneuraminic acid substrate, the enzyme will cleave the
chromogenic or fluorometric group from the substrate and the
reaction product will indicate the presence of the virion. The
enzyme, in the case of influenza is part of the virus itself, but
in other cases may be produced by the virus or by cells as a direct
result of the cells being infected with the particular virus.
An assay for the direct measurement of influenza neuraminidase was
developed by Yolken et al., J. Infectious Diseases 142:516-523
(1980). Yolken used the 4-methylumbelliferyl-.alpha.-ketoside of
N-acetylneuraminic acid as a fluorescent substrate to measure
neuraminidase activity in preparations containing small quantities
of cultivated influenza virus as well as in some nasal wash
specimens from human volunteers experimentally infected with
influenza virus. Yolken suggested that "successful development of
fluorometric enzyme assays for the detection of influenza
neuraminidase might thus provide for a practical means of influenza
diagnosis that is sufficiently rapid to allow for the institution
of appropriate preventative and therapeutic interventions". Id. at
522. Yolken attempted detection with colorimetric neuraminidase
assays. According to Yolken, the colorimetric assays were
insufficiently sensitive for clinical applications. In fact,
"visual neuraminidase substrates did not detect neuraminidase in
any nasal wash specimen" that Yolken studied. Id. at 520. In
contrast, Yolken noted that fluorometric assays may be suitable for
detecting influenza neuraminidase in clinical samples. "The
successful development of fluorometric enzyme assays for the
detection of influenza neuraminidase might thus provide for a
practical means of influenza diagnosis that is sufficiently rapid
to allow for the institution of appropriate preventative and
therapeutic interventions." Id. at 522. Following up on this
hypothesis, Pachucki et al., J. Clinical Microbiology 26:2664-666
(1988), tested the 4-methylumbelliferyl-.alpha.-ketoside of
N-acetylneuraminic acid on clinical specimens collected from
influenza patients. They reported that, due to its low sensitivity,
the "assay was not useful in detecting neuraminidase directly in
clinical specimens but identified 91% of virus-positive isolates 24
h after inoculation onto tissue culture". Id. at 2665.
The combined teachings of these references, therefore, lead away
from the use of neuraminidase substrates to detect influenza
neuraminidase activity in clinical samples, and further teach that
visual detection is impractical for any specimen.
SUMMARY OF THE INVENTION
One aspect of the invention is a method of detecting the presence
of a virus that comprises a characteristic enzyme in a clinical
sample suspected of containing the virus comprising the steps
of:
(a) incubating the sample with a solution of a substrate for the
enzyme, said substrate comprising a reactive group that is
liberated by reaction between the enzyme and the substrate and said
solution containing an agent that reacts with the liberated
reactive group to produce a visible reaction product, said
incubation being carried out under conditions that permit the
enzyme to react with the substrate to liberate the reactive group
and the reactive group to react with the agent; and
(b) visually observing the product of step (a) to determine the
presence or absence of said visible reaction product.
Another aspect of the invention is a method of detecting the
presence of a virus that comprises a characteristic enzyme in a
clinical sample suspected of containing the virus comprising the
steps of:
(a) incubating the clinical sample with a solution of a chromogenic
substrate for the enzyme and a precipitating agent, the chromogenic
group of the substrate exhibiting a characteristic color when
liberated from the substrate, said incubation being carried out
under conditions that permit the enzyme to react with the substrate
to liberate the chromogen and the liberated chromogen to be
precipitated by the precipitating agent;
(b) concentrating the precipitated chromogen; and
(c) visually observing the concentrate of step (b) for the presence
of said color.
Yet another aspect of the invention is a kit for use in the
detection of the presence of a virus that comprises a
characteristic enzyme in a clinical sample suspected of containing
the virus, said kit comprising in packaged combination:
(a) a buffer solution for extraction of the clinical sample;
(b) a substrate for the enzyme, said substrate comprising a
chromogen that is liberated by reaction between the enzyme and the
substrate; and
(c) a precipitating agent that in solution reacts with the
chromogen to produce a visibly detectable colored precipitate.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 generally illustrates an assay or test kit of the present
invention.
FIGS. 2A and 2B generally illustrates a means for concentrating the
precipitate. FIG. 2A provides a side view and FIG. 2B provides a
top view of the concentrating means.
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be used to detect the presence of any
virus that has a characteristic enzyme. Examples of such viruses
and enzymes are influenza A or B virus (neuraminidase, RNA
polymerase, ribonuclease), adenovirus (protein kinase, protease
endonuclease), CMV (ornithine decarboxylase, DNA polymerase,
protein kinase, exonuclease), herpes simplex (DNA polymerase,
protein kinase, thymidine kinase), parainfluenza
(hemagglutinin-neuraminidase), and respiratory syncytial viruses
(polymerase).
Substrates for these enzymes are known in the art, e.g.,
N-acetylneuraminic acid for neuraminidase, thymidyl phosphate for
thymidine kinase, adenylphosphate for protein kinase, and ornithine
for ornithine decarboxylase. These substrates may be coupled to
chromogens by known chemistry. For instance, in the case of
N-acetylneuraminic acid, the acid is first converted to its methyl
ester by treatment in methanol with trifluoroacetic acid. Reaction
of the ester in excess acetyl chloride acetates the free alcohol
groups on the substrate and effects conversion to the glycosyl
chloride simultaneously. The glycosyl chloride in chloroform is
then reacted with an aqueous solution of the sodium salt of the
chromogen in the presence of a phase transfer reagent such as
triethylammonium chloride to effect coupling. Deprotection of the
acetate and methyl ester groups by treatment with sodium methoxide
and sodium hydroxide yields the chromogen/N-acylneuraminic acid
conjugate.
The clinical samples that are tested in the invention will
typically be pharyngeal, nasopharyngeal (in the case of pediatric
patients) or respiratory secretions collected from patients as
wash, expectorate or swab specimens.
Proper collection and handling of clinical specimens is the most
important factor in a successful assay. The possibility of virus
isolation is increased when specimens are collected as soon as
possible (3 to 7 days) after disease onset.
In the case of influenza and depending on the type of respiratory
syndrome experienced by the patient, several different specimens
may be collected as outlined below.
Upper Respiratory Tract Infections
Colds are acute, self-limited viral infections of the epithelial
surfaces of the upper airway characterized by nasal discharge and
stuffiness, sneezing, rhinitis, and throat irritation. In infants,
additional symptoms may include obstruction of the nasal passages,
fever, and irritability. Low volume nasal washes (2 ml or less) and
nasal aspirates usually reveal high virus titers.
Pharyngitis is an inflammatory disease of the mucous membranes and
structures of the throat often involving the nasopharynx, uvula,
and soft palate. Soreness and irritation are involved, but rarely
is severe pain or difficulty in swallowing apparent. Additional
symptoms include fever, headache, nausea, vomiting, abdominal pain,
and cervical adenitis. Diagnosis requires objective evidence such
as erythema, exudate, or ulceration. Malaise, myalgia, chills,
dizziness, and cough are also occasionally noted. Pharyngeal swabs
or nasopharyngeal aspirates should be collected, especially if
nasal symptoms are prominent. Low volume throat washings or swabs
may be preferable if there is prominent pharyngitis.
Lower Respiratory Tract Infections
Influenza syndrome includes fever, chills, sore throat, and
systemic complaints. A cough is also associated with common cold
and pharyngeal symptoms. This syndrome is most often observed
during epidemics throughout the winter months. Throat swabs or
washings should be collected.
Pneumonia, and infiltrative disease of the lungs is most frequently
viral in nature for infants and children. Respiratory tract
symptoms include cough and shortness of breath. Nasopharyngeal
aspirates, tracheal aspirates, bronchial aspirates or nasal washes
are preferred. Throat swabs are acceptable.
Correct collection of the above-listed specimens will help to
assure a timely diagnosis. Specimen collection, processing, and
culture isolation of respiratory viruses should be attempted only
by experienced personnel. Nasopharyngeal swabs may not retrieve a
large number of virions, but are much easier to collect, especially
with infants. If secretions are too small as in the convalescent
stage of croup, or the mucosa particularly fragile, nasopharyngeal
swabs may be the specimen of choice. Throat swabs or washes may be
combined with nasopharyngeal swabs or collected alone particularly
with influenza virus infections. Nasal washes provide an easy
collection method, as with swab specimens, but increase the number
of mucosal cells available. Nasopharyngeal aspirates, particularly
where respiratory syncytial virus (RSV) and parainfluenza virus
infections are suspected, are strongly encouraged.
Nasopharyngeal swab specimens are collected as follows: A dry swab
(cotton or Dacron) is inserted into one or both nostrils to the
nasopharyngeal area. The swab is allowed to remain in the nostril
for a few seconds to absorb secretions, rotated gently, and then
withdrawn. A separate swab used for each nostril may increase the
specimen volume. Swab(s) should be placed into 1 to 2 ml of aqueous
buffer solution, the shaft broken off, and the cap tightly
secured.
Throat swab specimens are collected as follows: A swab (cotton or
Dacron), moistened in sterile phosphate buffered saline (0.15M
sodium chloride and 0.01M sodium phosphate, pH 7.0-7.6), is used to
vigorously rub the tonsils and the posterior pharynx. The swab is
then extracted into a sterile vial containing 2 ml of aqueous
buffer solution, and the vial is capped.
Throat wash specimens are collected as follows: The patient is
asked to gargle with 1 to 2 ml of sterile phosphate buffered saline
(0.15M sodium chloride and 0.01M sodium phosphate, pH 7.0-7.6) and
the buffered saline is collected into a sterile vial containing 1
to 2 ml of aqueous buffer solution and the cap tightly secured.
Nasal wash specimens are collected as follows: 1 to 2 ml of sterile
phosphate buffered saline (0.15M sodium chloride and 0.01M sodium
phosphate, pH 7.0-7.6) is instilled into each nostril while the
patient holds his/her head tilted back. Then the head is tipped
forward and the fluid collected into a sterile vial containing 1 to
2 ml of aqueous buffer solution and the cap tightly secured.
Nasopharyngeal wash specimens are collected as follows: 1 to 2 ml
of sterile phosphate buffered saline (0.15M sodium chloride and
0.01M sodium phosphate, pH 7.0-7.6) is aspirated into a soft rubber
bulb. The patient is placed on his/her side in a supine position
and one nostril is gently pressed closed with finger pressure. The
point of the bulb is used to completely occlude the other side. The
phosphate buffered saline is then squeezed into the nose and
quickly aspirated. Secretions are then expelled into a sterile vial
containing 1 to 2 ml of aqueous buffer solution and the cap tightly
secured.
Nasopharyngeal aspirate specimens are collected as follows: A No. 8
French soft plastic feeding tube is attached through a
valve-containing trap to an electric suction apparatus. The sterile
catheter is introduced through the flares to the back of the nose.
Suction is intermittently applied through the means of a thumb
valve while the catheter is slowly withdrawn. The process may be
repeated once in each nostril so that 0.2 to 0.8 ml of secretion is
obtained in the trap. The secretion is then transferred to 1 to 2
ml of aqueous buffer solution and the cap tightened securely.
The aqueous buffer solution referred to in the above collection
procedures preserves enzyme activity. It maintains the pH at about
4 to 7, preferably 5.5 to 6.5. The solution also contains
optionally about 0.1% to 10% by weight nonionic detergent, a small
amount (1-20 mM) of alkaline earth metal cation (Ca, Mg, preferably
Ca), and a sufficient amount of a stabilizer selected from the
group consisting of polyhydric sugar alcohols, simple sugars, and
disaccharide sugars to enhance the thermal stability of the enzyme
in the sample. In the case of washes and aspirates, if the specimen
is to be assayed within five minutes of collection, it may be
combined directly with the substrate and precipitating agent and
need not be combined with buffer solution. Similarly, fresh swabs
may be extracted directly into the substrate and precipitating
agent solution if they are to be tested within five minutes after
collection.
The buffer may be organic or inorganic. Examples of suitable
buffers are conventional buffers (e.g. monosodium citrate-disodium
citrate mixture, citric acid-trisodium citrate mixture, citric
acid-monosodium citrate mixture, etc.), acetate buffers (e.g.,
acetic acid-sodium acetate mixture), succinate buffers (e.g.
succinic acid-monosodium succinate mixture, succinic acid-sodium
hydroxide mixture, succinic acid-disodium succinate mixture, etc.),
tartrate buffers (e.g. tartaric acid-tartrate mixture, tartaric
acid-potassium tartrate mixture, tartaric acid-sodium hydroxide
mixture etc.), fumarate buffers (e.g. fumaric acid-monosodium
fumarate mixture, fumaric acid-disodium fumarate mixture,
monosodium fumaric acid-disodium fumarate mixture), gluconate
buffers (e.g. gluconic acid-sodium gluconate mixture, gluconic
acid-sodium hydroxide mixture, gluconic acid-potassium gluconate
mixture, etc.), oxalate buffers (e.g. oxalic acid-sodium oxalate
mixture, oxalic acid-sodium hydroxide mixture, oxalic
acid-potassium oxalate mixture, etc.), lactate buffers (e.g. lactic
acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture,
lactic acid-potassium lactate mixture, etc.), acetate buffers (e.g.
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.), malate buffers (e.g., D,L-malic acid-disodium
malate mixture), phosphate buffers (e.g. monosodium
phosphate-disodium phosphate mixture, monosodium phosphate-sodium
hydroxide mixture, trisodium phosphate-hydrochloric acid mixture,
etc.), 2-(N-morpho-lino)ethanesulfonic acid,
[bis-(2-hydroxyethyl)-imino]tris(hydroxymethyl)methane,
N-2-acetamido-iminodiacetic acid,
1,3-bis[tris(hydroxymethyl)methyl-amino]propane,
piperazine-N,N'-bis(2-ethanesulfonic acid),
N-2-acetamido-2-aminoethanesulfonic acid,
3-(N-morpholino)-2-hydroxypropanesulfonic acid,
N-N-bis-(2-hydroxyethyl)2-aminoethanesulfonic acid,
3-(N-morpho-lino)propanesulfonic acid,
2-[tris(hydroxymethyl)methyl-amino]ethanesulfonic acid,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid,
3-{[tris-(hydroxymethyl)-methyl]amino}-2-hydroxypropanesulfonic
acid.
Examples of non-ionic detergents useful in the buffer solution are
the Pluronics such as Polysorbate 20 and Polysorbate 80, Triton
X-100, NP-40, and alkyl glucosides such as C.sub.8 -C.sub.9 alkyl
glucoside. The detergent is an optional component and facilitates
release of the enzyme from the viral envelope.
Examples of the stabilizers that are used in the buffer solution
are trihydric or higher sugar alcohols, such as glycerin,
erythritol, arabitol, xylitol, sorbitol, mannitol, the simple
sugars glucose and fructose and the disaccharide sucrose. These
polyhydric sugar alcohols, and simple and disaccharide sugars can
be used alone or in combination. In order to stabilize the activity
of the neuraminidase-containing viruses, the polyhydric sugar
alcohols or simple and disaccharide sugars are added to the liquid
formulation/excipient system in an amount from 0.2M to 2.1M and
preferably, 0.6M to 2.0M.
Once mixed with the buffer solution, the sample may be stored for
prolonged periods, preferably at 2.degree. C. to 8.degree. C.
without significant loss of enzyme activity. Repeated freezing and
thawing of the sample should be avoided.
The fresh specimens (used within five minutes of collection) or the
buffered stabilized specimen is then combined and incubated with a
mixture of chromogenic substrate and precipitating agent
appropriate to detect the virus via the enzyme associated with the
virus. The reaction is carried out between ambient and
physiological (approximately 20.degree.-40.degree. C.), preferably
at room temperature. Higher temperatures may be used, but they may
produce false positives. The sensitivity of the test at
physiological temperature is substantially greater than at ambient
temperatures. The reaction time will normally be in the range of 20
to 120 minutes, more usually 30 to 60 minutes. The chromogenic
substrate concentration will normally range from 0.05 mM to 5.0 mM,
preferably 0.05 mM to 0.3 mM. If there is enzyme activity in the
sample, the chromogenic group will be cleaved from the substrate
and the liberated chromogen will react with the precipitating agent
to form a precipitate having a characteristic color.
The agents that allow for precipitation and visual detection of the
chromogen may be salts, dyes or color-producing compounds that can
be in their activated or inactive state whereby upon activation
they will produce the precursor to the final colored compound.
These dyes or color-producing compounds include any compounds which
will couple to, or react with the liberated chromogen, but not
limited to the diazonium salts listed below. Diazonium salts are
formed by the reaction of a precursor amine with a nitrite. Thus,
the aryl amine precursors to diazonium salts may be used in
combination with an alkyl nitrite, e.g., n-butyl nitrite and
t-butyl nitrite, at an acid pH. For maximum stability the diazonium
salt and substrate are preferably maintained in a dry (e.g.,
lyophilized) form prior to use. The concentration of the diazonium
salt, dye, or color producing compound will typically range between
0.01 mM to 10 mM with optimum ranges between 0.05 mM to 0.5 mM.
Concentrations between 0.025 mM to 5 mM have been tested with
several diazonium salts and results indicate that with increasing
concentrations of these diazonium salts sensitivity can be
increased to an optimal point.
The preferred aryl alcohol chromogens and their characteristic
colors when detected visually or colorimetrically after
precipitation by a diazonium salt are listed in the following
table. The colors were determined by the interaction of 0.2 mM
indicated diazonium salt solution with a 0.2 mM free indicated
chromogen solution in a 50 mM sodium acetate buffer at pH 5.9 and
allowing the reaction to proceed for several minutes and collecting
the precipitate on a 0.45 micron nitrocellulose membrane.
__________________________________________________________________________
INTERACTING DIAZONIUM SALT Released* Fast Fast Red- Fast Fast Fast
Fast Fast Chromogen Black K Violet LB Red ITR Red AL Corinth V Blue
BB Violet B
__________________________________________________________________________
MOP brown yellow- sand burnt maroon mauve peach orange orange DMP
brown- red- peach dark dark red orange black orange peach purple
6BN dark red orange burnt dark purple maroon brown orange purple
3AAP tan yellow yellow peach dark sand yellow peach 3BP tan yellow
sand beige burnt none none orange 4CN black brown berry sienna
black brown- brown brown black CONTROL flesh none none ivory flesh
none none (no chromogen)
__________________________________________________________________________
*The released chromogens in solution at the indicated
concentrations produced no color. MOP = 3methoxyphenol; DMP =
3dimethylaminophenol; 6BN = 6bromo-2-naphthol 3AAP =
3acetamidophenol; 3BP = 3bromophenol; 4CN = 4chloro-1-naphthol.
In addition to the substrate and precipitating agent, the reaction
mixture may contain color enhancing agents and moieties required
for enzymatic activity. In this regard, thimerosol (at 0.1% to 0.5%
by weight) and methyl paraben or propyl paraben (at 0.01% by
weight) were found to enhance the color of the precipitate formed
from several of the above-listed diazonium malts. Calcium ion has
been found to greatly increase the enzyme turnover rate and is thus
a desirable component of the mixture. Calcium ion may be added as a
salt, e.g., calcium chloride, preferably at concentrations in the
range of 5-20 mM, preferably 10 mM. Uncoupled chromogens may be
included to provide contrasting color which would distinguish a
positive test from a negative test. Bulking or stabilizing agents
such as mannitol may also be added to the substrate and salt.
The volume of the sample used in the assay may affect the
sensitivity and the presence of background color. Assay volumes
ranging between 0.25 ml and 2.0 ml have been used successfully.
Concentrations of substrate and precipitating agent should be
adjusted to give optimum results for each assay volume. In this
regard, it was found that when the assay volume is increased (i.e.,
concentration of enzyme is decreased) the concentrations of
substrate and precipitating agent must be decreased to reduce
undesirable background color formation.
The liberation of chromogen and the formation of precipitate are pH
dependent, and the optimum pH for each of these reactions may
differ. Generally the pH may range between about 3 and 9,
preferably between 5 and 8. In tests using a panel of two
chromogens and three diazonium salts, the most intense color was
produced at pH 6.9.
It may be desirable to improve the detection limit afforded by the
present invention for various samples. In such a case, an enzyme
amplification technique described by Bobrow et al., Journal of
Immunological Methods, 125:279-285 (1989), may be useful. The
method involves "utilizing the ADRE (analyte dependent reporter
enzyme) of a solid-phase assay to catalyze the deposition of
additional reporter enzyme onto the solid phase, resulting in
signal amplification and improved assay detection limits". Id. at
280.
The colored precipitate that is produced may be concentrated by any
conventional liquid-solid separation means, such as filtration,
evaporation, and centrifugation, so that it may be more readily
seen. Preferably the reaction medium is filtered using a suitable
filter with a pore size small enough to retain the precipitate.
Pore sizes will typically range between 0.20 microns and 20
microns, preferably from 0.45 microns to 5 microns. Examples of
suitable filter materials are nylon, chemically modified nylon,
nitrocellulose and hybrid nitrocellulose. The reaction medium may
be passed through the filter by gravitation or under positive
force, such as that effected by positive pressure, vacuum or
capillary action. In the latter an absorbent porous material, such
as cotton or cellulose, is placed beneath and in contact with the
membrane. Such material is capable of wicking and retaining the
fluid volume in the reaction mixture. The reaction mixture may be
focused on a small spot on the membrane to provide a distinct
colored spot on the surface of the membrane indicating a positive
test. The sensitivity of the assay may be increased by focusing the
mixture through a small diameter spot so as to concentrate the
color in a small area. The spot diameter will normally range
between 1 and 5 mm.
The invention further provides a kit for use in the clinic or
physician's office for rapidly detecting the presence of a virus in
a clinical sample. A preferred embodiment kit comprises a vessel
that contains a buffer solution wherein the sample can be
deposited. The kit further contains a second sealed vessel that
contains the substrate coupled to the chromogen and the
precipitating agent in dry form. When the sample is deposited in
the buffer solution (or the buffer solution is used to dissolve the
substrate and precipitating agent), the contents of the sealed
vessel are added and the solution is covered and allowed to
incubate for a period as described previously. The cover for the
buffer vessel is a filtration device comprising a porous membrane
and absorbent material. The center portion contains a 2 cm diameter
circular opening such that when the device is placed over the
vessel, the vessel is sealed from the outside air. The filtration
device further comprises a focusing device (e.g., a funnel) such
that when the sample is deposited on the membrane by inversion of
the vessel and filtration device combination, the solution is
physically concentrated to a spot on the membrane which is between
1.5 and 5.0 mm. After the incubation period is completed, the
vessel and filtration device are inverted to permit the reaction
mixture to pass through the membrane and where virus is present, a
colored reaction product will be present on the spot surface of the
membrane.
The kit will also normally include positive and negative control
specimens and written instructions for carrying out the assay.
FIG. 1 generally illustrates one embodiment of the kit 10 of the
present invention, which kit 10 consists of a plurality of first
sealed vessels 12 containing a buffer solution and other liquid or
dissolved components, a plurality of second vessels 16 sealed with
caps 18 containing the substrate coupled to the chromogen and the
precipitating agent in dry form, a plurality of filtration devices
20, and a plurality of swabs 14 for collection of the clinical
samples. The kit may also contain an instruction booklet or other
printed information (not shown) describing the use of the kit. The
kit 10 shown in FIG. 1 has five each of the various components and
can, therefore, be used to analyze five clinical samples. FIG. 2
illustrates the filtration device 20 of FIG. 1 in more detail. FIG.
2A shows a side view and FIG. 2B a top view of device 20. This
filtration device 20 has a body 22 which contains a porous membrane
24, absorbent material 26, and a funneling device 30 to direct and
focus the incubated mixture to a controlled spot or location 32 on
the porous membrane 24.
In use, the contents of one vessel 12 are used to reconstitute the
dry reagents of one vessel 16. A clinical sample is collected using
a single swab 14 (or other appropriate collection means). The swab
14 (or other appropriate means) is then used to deposit or
introduce the clinical sample directly into the reconstituted
solution in vessel 16. The mixture in vessel 16 is then sealed with
cap 18 and allowed to incubate for the desired time period. The
sample could, of course, be incubated in any suitable vessel if
desired. After incubation is complete, cap 18 is removed and the
contents of vessel 16 are poured or otherwise deposited into the
funneling device 30 in filtration device 20 such that the porous
membrane 24 acts as a filter. Any colored precipitate formed is
collected on spot 32 of porous membrane 24 and the fluid is wicked
away by absorbent material 26. The presence or absence of a
particular colored precipitate at spot 32 is then used to indicated
the presence or absence of the particular virus. Thus, once the
incubated sample is introduced into the filtration device 20, the
funneling device 30 will force or guide the colored precipitate (if
formed) to collect on spot 32, thereby increasing the sensitivity
of the analysis.
As those skilled in the art will realize, other embodiments of the
kit 10 and the filtration device 20 can be employed in the practice
of this invention.
Accordingly, the present invention provides a simple and rapid
technique for detecting the presence of a virus in a clinical
specimen that may be carried out in the clinic or physician's
office and enable the physician to prescribe the appropriate
therapy to treat the infection and/or the appropriate prophylactic
treatment to persons in close contact with the infected
patient.
The following example further illustrates the invention. This
example is not intended to limit the invention in any manner.
EXAMPLE
Clinical specimens from patients suspected of having influenza are
collected as described above. The specimen is combined with an
aqueous solution of 2.0 ml containing 10 mM CaCl.sub.2, 3.5 mM
malic acid, pH 5.9 and 0.01% Thymerosol. The combination of the
specimen and aqueous solution are used to reconstitute a dry
reagent mixture containing 0.075 mM
4-chloro-1-naphthol-N-acetyl-neuraminic acid, 0.05 mM Fast Red ITR,
0.04 mM 3AAP, 1% mannitol, and 21.5 mM malic acid diazonium salt in
a sterile vial.
Except in the case of wash or aspirate specimens where 2 ml wash or
aspirate itself are added directly to the dry reagents mixture.
After combining the specimen with the substrate mixture, the vial
is capped and the mixture is incubated at 37.degree. C. for 60
minutes.
The entire contents of the vial are then placed on a nitrocellulose
filter (1.2 microns, Micron Separation, Inc.) backed with a pad of
cellulose acetate (4.9.times.29.2.times.30.7 mm). The contents are
focused onto the filter through a 2 mm diameter orifice.
The color of the 2 mm spot through which the contents have been
filtered is observed immediately after all the solution has been
absorbed through the filter. A red, pink, or orange colored spot is
positive: a yellow colored spot is negative.
Modifications of the above described modes for carrying out the
invention that are obvious to those of skill in the fields of
virology, biochemistry, organic chemistry, diagnostic medicine and
related fields are intended to be within the scope of the following
claims.
* * * * *